C-X-C domain ligand 14-mediated stromal cell–macrophage interaction as a therapeutic target for hand dermal fibrosis

Dupuytren’s contracture, a superficial dermal fibrosis, causes flexion contracture of the affected finger, impairing hand function. Specific single-nucleotide polymorphisms within genes in the Wnt signalling pathway are associated with the disease. However, the precise role of Wnt signalling dysregulation in the onset and progression of Dupuytren’s contracture remains unclear. Here, using a fibrosis mouse model and clinical samples of human Dupuytren’s contractures, we demonstrate that the activation of Wnt/β-catenin signalling in Tppp3-positive cells in the dermis of the paw is associated with the development of fibrosis. Fibrosis development and progression via Wnt/β-catenin signalling are closely related to stromal cell–macrophage interactions, and Wnt/β-catenin signalling activation in Tppp3-positive stromal cells causes M2 macrophage infiltration via chemokine Cxcl14, resulting in the formation of a TGF-β-expressing fibrotic niche. Inhibition of Cxcl14 mitigates fibrosis by decreasing macrophage infiltration. These findings suggest that Cxcl14-mediated stromal cell–macrophage interaction is a promising therapeutic target for Wnt/β-catenin-induced fibrosis.


D
upuytren's contracture is a superficial dermal fibrotic disease in the hand that causes flexion contracture of the affected fingers and impairs hand function.Genetic predisposition, ethnic characteristics, and other factors, including alcohol intake, smoking, diabetes, repetitive trauma, and exposure to vibration, have been identified as risk factors for Dupuytren's contracture development, suggesting that it is a multifactorial disease 1 .Dupuytren's contracture represents a prevalent condition, particularly among Western populations, with an estimated prevalence of 21% by the age of 65 years 2 .Although a collagenase Clostridium histolyticum injection is available as a minimally invasive therapy, surgical excision remains a reliable treatment method for this disease 3 .However, surgical complications, including hematomas, nerve injuries, and wound healing complications, are frequently observed, with incidences of 23% 4 .
Myofibroblasts, the predominant stromal cells in Dupuytren's contracture, play a central role in fibrosis by producing excessive extracellular matrix and pathological collagen 5 .Recent genomewide studies have identified single-nucleotide polymorphisms (SNPs) in a subset of genes involved in the Wnt signalling pathway, including WNT2, WNT4, WNT7B, SFRP4, RSPO2, and SULF1 6,7 .β-catenin, a downstream target of canonical Wnt signalling, is expressed in most myofibroblasts in the nodule, accumulating in their nuclei 8 .Therefore, the involvement of such Wnt signalling-related SNPs in Dupuytren's contracture has been investigated 7,[9][10][11] .The expression of WNT7B, a canonical Wnt ligand, was increased in Dupuytren's nodules compared with that in unaffected palmar aponeurosis 9,10 .Moreover, its risk genotype was associated with Dupuytren's contracture development 11 .The mRNA expression of SFRP4, a Wnt antagonist, was increased in nodules compared with that in unaffected palmar aponeurosis in Dupuytren's patients 7,9 .However, its protein secretion decreased in Dupuytren's tissues with a high-risk genotype compared with that in those with a low-risk genotype 7 .Thus, the Wnt signalling pathway is likely implicated in Dupuytren's contracture.
Recent studies revealed the possible involvement of stromal cell-immune cell interaction in myofibroblast differentiation and its activation in Dupuytren's contracture [12][13][14] .Mast cells and macrophages, especially alternatively activated macrophages (M2), are involved in the fibrotic mechanism via chemotactic factors [12][13][14] .However, the role of Wnt signalling activation in the development and progression of Dupuytren's contracture remains to be elucidated.Moreover, additional research is required to investigate the role of Wnt signalling in the interaction between stromal and immune cells.
The aim of this study was to investigate the relationship between Wnt signalling activation and the development of dermal fibrosis using a fibrosis mouse model and human clinical samples of Dupuytren's contractures.Our analyses revealed that activation of Wnt/β-catenin signalling in dermal tubulin polymerizationpromoting protein family member 3 (Tppp3)-positive cells resulted in the development of fibrosis of the paw, and its progression was markedly dependent on Wnt/β-catenin signalling activity.However, in addition to Wnt/β-catenin signallingdependent myofibroblast differentiation in a cell-autonomous manner, fibrosis development and progression via Wnt/β-catenin signalling were closely related to stromal cell-immune cell interactions marked by M2 macrophage infiltration.Furthermore, transforming growth factor-β (TGF-β) was highly expressed in the fibrotic niche, consisting of stromal cells and macrophages, causing myofibroblast differentiation of stromal cells.Notably, the activation of Wnt/β-catenin signalling in Tppp3-positive cells upregulated the expression of chemokine C-X-C domain ligand 14 (Cxcl14), which is involved in macrophage infiltration.Furthermore, the blockade of Cxcl14 mitigated Wnt/β-catenininduced fibrosis of the paw by decreasing macrophage infiltration.

Results
TPPP3 is expressed in the human palmar fascia and Dupuytren's contractures.A recent study analysed human clinical samples and reported that pericytes are the putative cellular origin of Dupuytren's contractures 15 ; however, their exact origin remains unknown 1 .Therefore, we investigated clinical samples of the human palmar fascia and Dupuytren's contracture to identify candidate markers of origin cells and establish a dermal fibrosis mouse model that mimics Dupuytren's contractures.
Dupuytren's contractures generally occur in the dermis of the palmar hand and continue to the palmar fascia.TPPP3 is a known paratenon and synovial cell marker around the tendon and fascia 16,17 .As expected, TPPP3 expression was detected in the cells on the surface of the palmar fascia (Fig. 1a).In addition, TPPP3 was expressed in human Dupuytren's contractures, with strong expression in the nodule (Fig. 1b-d, Supplementary Fig. 1).Immunohistochemical analyses revealed that TPPP3-positive cells with fibroblast-like morphology on the surface of the palmar fascia did not co-express β-catenin and αSMA.Conversely, most TPPP3-positive cells in the nodule co-expressed β-catenin and αSMA, a myofibroblast marker (Fig. 1e).These results suggest that TPPP3-positive palmar fascial cells in the dermis potentially represent one of the candidates for cellular origin of Dupuytren's contractures.
Activation of Wnt/β-catenin signalling in Tppp3-positive cells causes dermal fibrosis of the paws.We investigated the localization of Tppp3-positive cells in the paws using tamoxifenadministered Tppp3-CreER T2 /Rosa26-loxP-stop-loxP-tdTomato (Rosa26-stop-tdTomato) mice and confirmed that many tdTomato-positive cells were localized in the dermis of the paw (Fig. 2a, b).We created Tppp3-CreER T2 /Rosa26-stop-tdTomato/βcatenin ex3 flox mice and induced Wnt/β-catenin signalling activation in Tppp3-positive cells (Fig. 2c, Supplementary Fig. 2a-d).After tamoxifen administration, fibrotic regions comprising αSMA-expressing myofibroblasts were induced in the paw; however, it is noteworthy that the mice did not develop nodules and finger contractures analogous to those observed in human Dupuytren's contractures (Fig. 2d).Sequential histological analyses revealed that the dermal layer was thickened in a timedependent manner, although they were not palmar side-specific (Fig. 2e, Supplementary Fig. 2e).Dermal fibrotic region exhibited a significant increase in the number of myofibroblasts one month after Wnt/β-catenin signalling activation, followed by a subsequent reduction two months thereafter (Fig. 2f).
Next, we investigated whether myofibroblast phenotype and activity are directly regulated by Wnt/β-catenin signalling.Murine dermal fibrosis tissues derived from Dox-inducible βcatenin-expressing mice were harvested, cultured in vitro, and treated with or without Dox (Fig. 3d, Supplementary Fig. 3b-d).Gene expression analyses using real-time PCR revealed that Dox treatment upregulated the expression of Axin2, a target gene of βcatenin (Fig. 3e).In addition, Dox treatment significantly upregulated the expression of myofibroblast markers (Acta2, Tagln, and Myh11) 18 , type I collagen (Col1al), and type III collagen (Col3a1) (Fig. 3e).Conversely, in αSMA-expressing myofibroblasts derived from dermal fibrous tissues, Ki67 immunostaining showed that Wnt/β-catenin signalling activation slightly reduced cell proliferation (Fig. 3f, g), which is inconsistent with previous findings 19  results of the murine dermal fibrosis model, Wnt/β-catenin signalling could serve as a therapeutic target for human Dupuytren's contractures.Therefore, we investigated the effect of smallmolecule inhibitors of Wnt/β-catenin signalling on Dupuytren's contracture-derived cells in vitro.We used PNU-74654, which inhibits the interaction between β-catenin and T cell factor (TCF), and MSAB, which degrades β-catenin directly 20 .Neither inhibitor decreased the expression levels of myofibroblast markers (ACTA2, TAGLN, and MYH11), nor did they impact the expression of collagens (COL1A1 and COL3A1) (Supplementary Fig. 4a, b).Another inhibitor of Wnt/β-catenin signalling, XAV939, did not degrade β-catenin in human Dupuytren's contracture-derived cells (Supplementary Fig. 4c).In contrast to mouse data (Fig. 3e), activation of Wnt/β-catenin signalling via Wnt3A administration did not upregulate the expression of myofibroblast markers and collagens (Supplementary Fig. 4d).These findings indicate that the modulation of Wnt signalling may not directly alter the myofibroblast phenotype and collagen expression in human Dupuytren's contracture-derived cells.
Treatment of murine dermal myofibroblasts and human Dupuytren's contracture-derived cells with TGF-β1, as a known major inducer of fibrosis, significantly upregulated the expression of myofibroblast markers and collagens and induced αSMApositive myofibroblast differentiation (Fig. 4h, i, Supplementary Fig. 6a-c).In vitro experiments showed that Wnt/β-catenin signalling activation in murine dermal myofibroblasts upregulated the expression of Tgfb1, Tgfb2, and Tgfb3 (Fig. 4j).In contrast, its activation in human Dupuytren's contracturederived cells did not upregulate the expression of these genes (Supplementary Fig. 6d).These findings suggest that TGF-βexpressing niche formation may be closely associated with stromal cell-macrophage interaction rather than a cellautonomous mechanism that occurs via the activation of Wnt/ β-catenin signalling.Moreover, TGF-β1 induced the nuclear translocation of β-catenin (Supplementary Fig. 6e), indicating that a positive interaction between Wnt/β-catenin and TGF-β signalling may be related to dermal fibrosis development and progression.
Tppp3-positive cells secrete Cxcl14 via Wnt/β-catenin signalling activation.We sought to investigate the mechanism by which Wnt/β-catenin signalling activation in Tppp3-positive cells leads to macrophage infiltration.Macrophage differentiation and recruitment are induced by chemotactic factors, including cytokines and chemokines 23 .In our fibrosis model, infiltrating macrophages were predominantly of the M2 subtype; therefore, we examined the expression of chemokines associated with M2 macrophage differentiation and recruitment.Cxcl14 expression was significantly upregulated in Dox-treated murine dermal fibrotic cells in vitro (Fig. 6a), and Cxcl14 expression was Fig. 2 Wnt signalling activation in Tppp3-expressing cells causes dermal fibrosis of the paw.a A schematic of the tamoxifen injection protocol for Tppp3-CreER T2 /Rosa26-stop-tdTomato(R26-tdT) mice.At 1 month of age, the mice were injected with 1 mg tamoxifen three times and analysed 7 days after the last injection.b Fluorescent immunohistochemistry of αSMA (green) and tdTomato (red) in the palmar side of the paw in Tppp3-CreER T2 /Rosa26-tdT mice.Several tdTomato-positive cells existed in the dermal layer of the paw.Dotted lines indicate the border of the dermis.c A schematic representation of the in vivo experimental setup involving Tppp3-CreER T2 /R26-tdT/β-catenin ex3 flox mice.Notably, activated β-catenin without phosphorylation sites in exon 3 can be induced in Tppp3-positive cells via tamoxifen injection.At 1 month of age, the mice were injected with 1 mg of tamoxifen or corn oil (control) and analysed 1 and 3 months after the injections.d Fluorescent immunohistochemistry of murine dermal fibrotic lesions in the paw 1 month after tamoxifen injections.βcatenin activation in the dermal Tppp3-lineage cells (tdTomato + ) caused fibrosis, as indicated by αSMA expression.Dotted lines indicate the border of the dermis.e H&E staining and Masson's trichrome staining of Tppp3-CreER T2 /R26-tdT/β-catenin ex3 flox mice treated with corn oil or tamoxifen 1 and 3 months after injections.Compared to the corn oil groups (left panels), the tamoxifen groups (right panels) exhibited a notable and statistically significant increase in dermal thickness on the palmar side of the paw at both 1 and 3 months (p = 0.0160 and 0.0289, respectively).The graph illustrates the relative dermal thickness, with the mean dermal thickness in each sample of the corn-oil-treated group at 1 month set to 1.The mean ± SD of five and four independent biological samples (six different lesions per sample) are shown (two-tailed Mann-Whitney U test).Arrows in Masson's trichrome staining indicate the thickness of the dermis.f Fluorescent immunohistochemistry of the palmar side of the paws in Tppp3-CreER T2 /R26-tdT/β-catenin ex3 flox mice treated with corn oil (lefts) or tamoxifen (rights) 1 and 3 months after injections.Myofibroblasts were defined as αSMA (green)-positive/CD31 (red)-negative cells.The tamoxifen group showed significantly increased dermal myofibroblasts in the palmar side of the paw at 1 month (p = 0.0047); at 3 months, the increase was not significant (p = 0.1407).The graph indicates the relative number of myofibroblasts per area.The mean count of myofibroblasts within each sample from the corn oil treatment group at 1 month was set to 1.The mean ± SD of four independent biological samples (two different lesions per sample) are shown (two-tailed Mann-Whitney U test).Scale bars represent 50 µm (b, d, e, f).
increased in murine dermal fibrotic regions induced by Wnt/βcatenin signalling activation in vivo (Fig. 6b, c).Similarly, CXCL14 expression was detected in human Dupuytren's contracture samples (Supplementary Fig. 8a, b).CXCL14 expression increased in β-catenin-expressing cells in the nodule compared with that in non-diseased cells in the palmar fascia (Supplementary Fig. 8c, d), although it was detected in both αSMA-positive and -negative stromal cells in the nodule (Supplementary Fig. 8e).In addition, Wnt3A treatment upregulated CXCL14 expression in human Dupuytren's contracture-derived cells in vitro (Supplementary Fig. 8f, g).

A x i n 2 A c t a 2 T a g l n M y h 1 1 C o l 1 a 1 C o l 3 a 1
Anti-Cxcl14 neutralizing antibody treatment partially suppresses dermal fibrosis by reducing macrophage infiltration in mice.Finally, we investigated whether blockade of Cxcl14 in Wnt/β-catenin signalling in a fibrotic mouse model can suppress dermal fibrosis by reducing macrophage infiltration.Tppp3-CreER T2 /β-catenin ex3 flox mice administered tamoxifen were treated with an anti-Cxcl14 neutralizing antibody or control IgG for 1 month, and dermal fibrotic lesions of the paws were investigated (Fig. 7a).As expected, anti-Cxcl14 neutralizing antibody treatment decreased CD206-expressing M2 macrophage infiltration into the dermis (Fig. 7b).Compared with the corn oil treatment, the control IgG treatment showed significant thickening of the dermis on the paw, whereas the anti-Cxcl14 neutralizing antibody treatment partially suppressed Wnt/β-catenininduced dermal thickening of the paw (Fig. 7c).We found fewer αSMA-expressing myofibroblasts in mice treated with the anti-Cxcl14 neutralizing antibody than in those treated with the control IgG (Fig. 7d).These findings imply that neutralization of Cxcl14 partially suppresses Wnt/β-catenin signalling-induced fibrosis by inhibiting M2 macrophage infiltration.

Discussion
The cellular origin of Dupuytren's contracture and the significance of Wnt signalling activation in this disease remain elusive.In this study, we found that activation of Wnt/β-catenin signalling in Tppp3-positive cells in the dermis of the paw caused dermal fibrosis resembling human Dupuytren's contractures.Moreover, the development and progression of the disease were shown to involve the following: (1) a cell-autonomous fibrotic program induced by Wnt/β-catenin signalling activation in Tppp3-positive cells; and (2) a non-cell-autonomous fibrotic program mediated by TGF-β, which was released based on the interaction between Tppp3-positive stromal cells and macrophages via the chemokine Cxcl14, induced by Wnt/β-catenin signalling activation in Tppp3-positive cells (Fig. 8).Wnt/β-catenin signalling is an important pathway in organ development and cellular differentiation 24 .Dysregulation of Wnt/ β-catenin is involved in fibrosis in various organs and tissues in mice, rats, and humans 19,25,26 .Wnt/β-catenin signalling activation is associated with cell proliferation and migration and increased production of collagen and matrix 19,27,28 .Furthermore, it directly induces myofibroblast differentiation 19 .Conditional knockout of β-catenin and administration of Wnt/β-catenin signalling inhibitors (ICG-001, PRI-724) ameliorated bleomycininduced murine dermal and lung fibrosis and chronic hepatitis C virus-induced liver fibrosis in vivo [29][30][31][32] .In contrast, some studies have reported that the expression of αSMA, a typical myofibroblast marker, was not directly induced by activation of Wnt/βcatenin signalling via Wnt3A 28,33,34 .Our study showed that the myofibroblast phenotype was directly induced by Wnt/β-catenin Fig. 3 The development and progression of dermal fibrosis in mice are dependent on Wnt signalling activity.a A schematic representation of the in vivo experiment conducted on doxycycline (Dox)-inducible β-catenin-expressing mice (Tppp3-CreER T2 /Rosa26-stop-tdTomato/Rosa26-stop-rtTA/Col1a1::TetO-βcatenin).At 1 month of age, the mice were injected with 1 mg of tamoxifen three times, followed by Dox treatment (50 μg/mL).The upper panel shows the mice treated with or without Dox for 1 month.The lower panel showcases two groups: mice treated with Dox for 2 months and those initially treated with Dox for 1 month, followed by a one-month withdrawal of treatment.b Masson's trichrome staining of dermal fibrotic lesions in the paws of Dox-inducible βcatenin-expressing mice (Fig. 3a).Compared with the Dox-off 1 month group (control), the Dox treatment groups showed increased dermal thickness in a time-dependent manner (Dox-on 1 month group; p = 0.0123, and Dox-on 2 months group; p < 0.0010).Conversely, when compared with the Dox-on 1 month group, the Dox withdrawal group showed halted progression of dermal thickness (Dox-on 1 month + off 1 month group; p = 0.8961).The graph shows the relative dermal thickness, with the mean dermal thickness in each sample from the Dox-off 1 month group normalized to 1.The data are presented as the mean ± SD (one-way ANOVA with the Tukey-Kramer multiple comparison test).Biological samples consisted of Dox-off and -on 1 month (n = 8) and Dox-on 2 months and -on 1 month + off 1 month (n = 6) groups, with dermal thickness defined as the mean measurement across six lesions per sample.c Fluorescent immunohistochemistry was performed on dermal fibrotic lesions in the paws of the mice (Fig. 3a).Continuous Dox treatment was associated with the presence of αSMA-expressing fibrotic lesions in the dermis, whereas Dox withdrawal decreased the number of αSMA-expressing myofibroblasts in the dermis.The graph indicates relative myofibroblast counts in the dermis.The data are presented as the mean ± SD (one-way ANOVA with the Tukey-Kramer multiple comparison test).The mean relative myofibroblast count of the Dox-off 1-month group was set to 1. Compared with that in the Dox-off 1 month group, the relative number of myofibroblasts increased in the Dox-on 1 month group significantly (p = 0.0216).However, this increase was not statistically significant in the Dox-on 2 months group (p = 0.1803).Dox withdrawal (Dox-on 1 month + off 1 month group) decreased the relative number of myofibroblasts, albeit not significantly (p = 0.1262).Biological samples encompassed both Dox-off and -on 1 month (n = 8) and Dox-on 2 months and -on 1 month + off 1 month (n = 6) groups.Two different lesions were analysed per sample.Myofibroblasts were defined as αSMA-positive/ CD31-negative cells.d A schematic representation of the cell isolation protocol of dermal fibrotic tissues in the forepaws of Tppp3-CreER T2 /Rosa26-stop-rtTA/Col1a1::TetO-β-catenin mice.Fibrotic cells were cultured with Dox-containing media (0.2 µg/mL) and expanded for 2-3 weeks.e RT-qPCR results show the expression of the β-catenin target gene (Axin2), myofibroblast markers (Acta2, Tagln, and Myh11), and fibrotic collagens (Col1a1 and Col3a1).Isolated dermal fibrotic cells (Fig. 3d) were split into two groups: one with continued Dox treatment (Dox-on group) and one with Dox withdrawal (Dox-off group) for 24 h.The expression level of the Dox-off group in each sample was set to 1.The mean ± SD of seven independent biological samples (three technical replicates per sample) are shown (two-tailed paired t test).f Ki67-positive cell detection using immunocytochemical staining of dermal fibrotic cells (Fig. 3d).Ki67 (green) and αSMA (red).Upper: Dox-off; lower: Dox-on.g The Ki67-and αSMA-positive cell ratio of Dox-off and Dox-on groups in Fig. 3f.The mean ± SD values of two independent experiments with three independent biological samples are shown.The Dox-on group showed a significant decrease in the Ki67-positive cell ratio compared with the Dox-off group (Mann-Whitney U test; p = 0.0411).The αSMA-positive cell ratio showed no significant difference between the Dox-off and Dox-on groups (two-tailed Mann-Whitney U test; p = 0.6991).Scale bars represent 50 µm (b, c) and 100 µm (f).
signalling in mice but not in humans.Verjee et al. 13   TGF-β is closely associated with fibrosis and is a representative fibrosis inducer 35 .All three TGF-β isoforms induce a fibrotic response, although the role of TGF-β3 as a fibrosis inducer or repressor remains controversial 36 .Interestingly, TGF-β stimulated Wnt signalling by inducing the nuclear accumulation of βcatenin in rat pulmonary alveolar cells and human fibroblasts 37,38 and induced αSMA expression by interacting with Smad3, CBP, and β-catenin 38 .Therefore, β-catenin is essential for TGF-βmediated fibrosis.Similarly, we showed that TGF-β1 treatment induced nuclear translocation of β-catenin in human Dupuytren's contracture-derived cells.Myofibroblast differentiation via Wnt3A was observed in the presence of TGF-β 34 .Our in vivo analysis using Tppp3-CreER T2 /β-catenin ex3 flox /Tgfbr2 flox/flox (Tgfbr2-KO) mice demonstrated that TGF-β signalling was associated with Wnt/β-catenin-induced fibrosis.Together, these findings suggest that Wnt/β-catenin signalling and TGF-β signalling are intimately related to fibrosis and could be potential therapeutic targets for fibrotic disease.However, in this study, Wnt signal inhibitors PNU-74654, MSAB, and XAV939 did not reduce the expression of myofibroblast markers (including αSMA) and collagen in human Dupuytren's contractures.Moreover, using TGF-β inhibitors for molecular therapy is challenging owing to the crucial role of TGF-β in the maintenance of cell homeostasis 22 .Thus, targeting these signals directly in the treatment of Dupuytren's contractures may prove difficult.
Recently, the importance of stromal cell-immune cell interactions has been recognized in fibrotic disease development and progression 23,39 .Chronic inflammation by immune cells (particularly macrophages) is involved in fibrosis development in several organs and tissues, including pulmonary fibrosis, skin fibrosis, and liver fibrosis 23,39-41 .Dupuytren's contractures share this pathological process of the development and progression of fibrosis [12][13][14] .Izadi et al. 12 reported reciprocal activating pathways through stromal cells-expressed IL-33 and TNF-expressing M2 macrophages and mast cells.Furthermore, they demonstrated that anti-IL-33 and TNF receptor II antibody treatment downregulated the expression of fibrotic genes and reduced cellular contractility of Dupuytren's myofibroblasts.Akbar et al. 14 showed that mast cell-expressed IL-13 activated myofibroblasts through STAT signalling and that tofacitinib, an inhibitor of JAK1 and JAK3 related to the phosphorylation of STAT1 and STAT6, reduced proliferation and fibrotic gene expression in Dupuytren's myofibroblasts.Thus, it is expected that the stromal cell-immune cell interaction can serve as a novel therapeutic target for Dupuytren's disease.
In this study, we found that Wnt/β-catenin signalling activation in Tppp3-positive cells recruited macrophages and formed a TGF-β-expressing niche via Cxcl14.Macrophages are classified into two subtypes: classically activated macrophages (M1) and alternatively activated macrophages (M2).TGF-β is predominantly expressed in M2 macrophages, and its expression in these macrophages increases when they are in contact with fibroblasts 42 .Macrophage differentiation and recruitment are induced by several kinds of cytokines and chemokines [43][44][45][46][47] .Cxcl14/CXCL14 is the most recently identified C-X-C chemokine and is highly conserved in mice and humans 48 .Cereijo et al. 43 and Wang et al. 47 demonstrated that Cxcl14, secreted by brown adipocytes and pericytes, recruits macrophages and differentiates them into the M2 subtype, indicating that it could be an inducer of M2 macrophage polarization.Here, we demonstrated that Cxcl14/CXCL14 expression was upregulated via Wnt/β-catenin activation in murine dermal Tppp3-positive cells and human Dupuytren's contracture-derived cells.In addition, TGF-β1 upregulated Cxcl14/CXCL14 expression in murine dermal fibrotic cells and human Dupuytren's contracture-derived cells.Thus, a TGF-β-expressing niche, induced by Wnt/β-catenin signalling activation, may be stabilized via a positive-feedback interaction between stromal cells and macrophages through Cxcl14/CXCL14.
Cxcl14 is involved in the progression of murine liver fibrosis 49 .However, no study thus far has analysed its function in disease development and progression.Notably, knockdown of Cxcl14 Fig. 4 Wnt signalling activation in Tppp3-expressing cells leads to the formation of a TGF-β-expressing niche through macrophage infiltration.a Fluorescent immunohistochemistry of a dermal fibrotic region in the paw of Tppp3-CreER T2 /Rosa26-stop-tdTomato/Rosa26-stop-rtTA/Col1a1::TetO-βcatenin mice (Dox-inducible β-catenin mice).tdTomato-positive (white arrowheads) and -negative (yellow arrowheads) αSMA-expressing myofibroblasts were observed.b Fluorescent immunohistochemistry of the dermal fibrotic region in the paws of the same Dox-inducible β-catenin mice (Fig. 4a).CD68expressing macrophages infiltrated around tdTomato-positive cells (β-catenin-expressing Tppp3-positive cells).c Fluorescent immunohistochemistry showing the macrophage subtypes infiltrating the fibrotic region in Tppp3-CreER T2 /β-catenin ex3 flox mice.CD68 (red); pan-macrophage marker, CD206 (green); M2 macrophage marker.An average of 65% of the macrophages comprised M2 macrophages (yellow).The white square area is enlarged in the lower left panel.d Quantification of the number of CD206-expressing cells in the dermis of Tppp3-CreER T2 /β-catenin ex3 flox mice treated with corn oil or tamoxifen.Activation of β-catenin in Tppp3-positive cells significantly increased M2 macrophage infiltration in the dermis (two-tailed Mann-Whitney U test; p = 0.0087).The number of CD206-positive cells was counted in two random fields per sample.The means ± SD of three independent biological samples are shown.e Gene expression analyses using real-time PCR of M1 and M2 macrophages.RAW267.4 cells were differentiated into M1 macrophages with lipopolysaccharide (60 ng/mL) and M2 macrophages with interleukin-4 (40 ng/mL) for 30 h.Nos2 and Tnfa are M1 macrophage markers, whereas Mrc1 (CD206) and Arg1 are M2 macrophage markers.M2 macrophages expressed significantly higher Tgfb1-3 than M1 macrophages (p = 0.0046, 0.0477, and 0.0277, respectively).The mean ± SD values of three independent experiments (three technical replicates per experiment) are shown (two-tailed Mann-Whitney U test).f Fluorescent immunohistochemistry of the same region in Fig. 4c shows the TGF-β (red)-expressing niche with M2 macrophage (CD206; green) infiltration.Both M2 macrophages and their surrounding cells expressed TGF-β.The white square area is enlarged in the upper right panel.g Fluorescent immunohistochemistry of the fibrotic region in Tppp3-CreER T2 /β-catenin ex3 flox mice.A part of Tppp3-expressing stromal cells (green) also expressed TGF-β (red).The white square area is enlarged in the upper left panel.h Gene expression analyses using real-time PCR of TGF-β1 (10 ng/mL)-treated murine dermal fibrotic cells derived from Tppp3-CreER T2 /β-catenin ex3 flox mice in vitro.The mean ± SD values of five independent experiments (three technical replicates per experiment) are shown (two-tailed Mann-Whitney U test).i αSMA-positive cell detection by immunocytochemical staining of murine dermal fibrotic cells derived from Tppp3-CreER T2 /β-catenin ex3 flox mice.Smad2/3 (green) and αSMA (red).Upper: control (-TGF-β1) group; lower: 10 ng/mL of TGF-β1 treatment (+TGF-β1) group.The αSMA-positive cell ratio was significantly higher in the TGF-β1 treatment group than that in the control group.The mean ± SD values of two fields for three independent experiments are shown (two-tailed Mann-Whitney U test; p = 0.0411).j Tgfb1, Tgfb2, and Tgfb3 expression in murine dermal fibrotic cells derived from Dox-inducible β-catenin mice (Fig. 3d) was analysed using RT-qPCR.Significant differences were observed between the Dox-off and Dox-on groups (p = 0.0353, 0.0394, and 0.0179, respectively).The mean ± SD values of six independent biological samples (three technical replicates per sample) are shown (two-tailed paired t test).Scale bars represent 50 µm (a, b, c, f, g) and 100 µm (i).
expression and neutralization of Cxcl14 via antibodies ablated macrophage migration caused by the senescence-associated secretory phenotype both in vitro and in vivo, respectively 50 .Consistent with this report, we revealed that the neutralization of Cxcl14 in Tppp3-CreER T2 /β-catenin ex3 flox mice reduced M2 macrophage infiltration and mitigated dermal fibrosis in vivo.Moreover, CXCL14 is highly expressed in human idiopathic lung fibrosis 51 , and its expression was observed in Dupuytren's contractures 52 , suggesting that it may also be associated with macrophage infiltration in human diseases.
This study has some limitations.First, we generated a mouse fibrosis model with β-catenin gene mutations; however, β-catenin gene mutations are absent in human Dupuytren's contractures 8,53 .This limitation may have been responsible for the differences in myofibroblast differentiation and induction of TGF-β expression in response to Wnt/β-catenin signalling activation between mice and humans in this study.Therefore, to analyze the development and progression of Dupuytren's contracture smore accurately, it may be necessary to create alternative mouse models, focusing on known SNPs in Dupuytren's contractures 6,7 .
Second, Cxcl14 inhibition did not completely prevent M2 macrophage infiltration and fibrosis development; therefore, other factors controlling stromal cell-immune cell interaction could be involved in Wnt/β-catenin-induced fibrosis.Furthermore, Wnt/βcatenin signalling-induced fibrosis involves both cell-and noncell-autonomous fibrosis programs (Figs. 5, 7, and 8).Therefore, it becomes apparent that exclusively targeting Cxcl14 may not yield a marked therapeutic effect in vivo.
Third, we did not elucidate the precise mechanism through which Wnt/β-catenin signalling regulates Cxcl14/CXCL14 expression in Tppp3-positive cells and human Dupuytren's contracture-derived cells.Specifically, luciferase reporter constructs containing sequences spanning from +1908 to −292 relative to the start codon of murine Cxcl14 failed to upregulate promoter activity upon Dox-inducible β-catenin expression and TGF-β1 administration (see Supporting Data).These results suggest the possible existence of other regulatory mechanisms influenced by β-catenin and the TCF/LEF complex or suggest indirect regulation mediated by β-catenin-target genes.Moreover, we did not reveal the precise mechanisms by which Cxcl14 regulates TGF-β expression in M2 macrophages.Additionally, most of its receptors, with the exception of CXCR4 and IGF-1R 54,55 , remain elusive 48 , leaving the exact downstream pathway unclear.Further analyses are required to investigate the clinical application of Cxcl14-associated pathway-targeted therapy.Fourth, Cxcl14 may play a role in the regulation of the central nervous system, specifically in feeding behavior and host defense against infections 56,57 .In addition, Cxcl14-deficient mice exhibited lower weight, high blood glucose levels due to insulin resistance, and reduced bacterial clearance compared with wild-type mice, although the mice were viable 43,58 .Therefore, we should validate the risk of side effects due to blocking Cxcl14 and overcome them for clinical application.
Collectively, identifying Cxcl14-mediated cellular interaction between stromal cells and macrophages via activation of Wnt/βcatenin signalling and the formation of a TGF-β niche reflects an important process in the development and progression of fibrotic diseases.This mechanism of cellular crosstalk between stromal cells and macrophages could be a promising therapeutic target for fibrosis, including Dupuytren's contracture.

Methods
Human participants.Experiments using clinical samples of human Dupuytren's contracture were approved by the Ethics Committee of Gifu University (approval number 28-140).
Surgically resected human Dupuytren's contracture tissues were obtained.From these tissues, nodular regions were shredded into 2-3 mm pieces and dissociated with collagenase for 30 min at 37 °C.Next, they were cultured in a 10 cm dish (Thermo Fisher Scientific) with DMEM supplemented with 10% FBS and 1% penicillin/streptomycin for approximately 2 weeks.
Migration and chemotaxis assay.The migration and chemotaxis assay were performed as previously described 43,47 .For the migration assay, the macrophages were cultured on 12-well plates.At 100% confluence, they were treated with RPMI-1640 medium (Thermo Fisher Scientific) and supplemented with or without 10 nM recombinant mouse CXCL14/BRAK protein (R&D Systems).The bottom of the plates was scratched with a 200 µL micropipette tip and analysed at 0 h and 30 h after scratching.
For the chemotaxis assay shown in Figs.6e, 1.5 × 10 5 (200 µL) macrophage cells were placed into Costar Transwell chambers on 24-well plates (Corning Incorporated, NY, USA).The bottom wells of the plates were filled with serum-free medium, with or without 10 nM recombinant mouse CXCL14/BRAK protein (R&D Systems).Afterward, the plates were incubated at 37 °C for 30 h.In the chemotaxis assay depicted in Fig. 6g, a  conditioned medium alone, and conditioned medium with 20 µg/ mL of a control IgG (AB-108-C; R&D Systems) or anti-CXCL14 neutralizing antibody (AF866; R&D Systems) 50 .A supernatant culture medium of murine dermal fibrotic cells derived from Tppp3-CreER T2 /β-catenin ex3 flox mice was used as the conditioned medium.The plates were subsequently incubated at 37 °C for 24 h.The transwell chamber containing macrophages was washed with PBS, and the cells were fixed with 2% paraformaldehyde (PFA) for 20 min and stained with 0.2% crystal violet for 5 min at room temperature.Non-migrating cells on the upper side of the membranes were removed by scraping.The membranes were attached to glass slides.Finally, migrating macrophages were counted using a microscope (BX51, Olympus, Tokyo, Japan).
Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR).RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN).Up to 1 µg of RNA was used for reverse transcription into cDNA using the PrimeScript RT reagent kit (TAKARA).Real-time quantitative RT-PCR (RT-qPCR) was performed using Premix Ex Taq™ (Perfect Real Time) (TAKARA).Transcript levels were analysed with three technical replicates and normalized to those of ACTB/Actb.The primer sequences are listed in Supplementary Table S1.The secondary antibody for 3, 3′-diaminobenzidine staining was Dako EnVision (Dako Japan, Inc., Kyoto, Japan).The samples were incubated at room temperature for 30 min, and the stained cells were analysed using microscopy (BX51, Olympus).For immunofluorescence, the secondary antibodies were conjugated with Alexa Fluor 488 and Alexa Fluor 594 (Thermo Fisher Scientific), followed by incubation with the cells at room temperature for 2 h.Nuclei were counterstained with DAPI (Cell Signaling Technology).The stained cells were analysed using fluorescence microscopy (IX83, Olympus).
Western blot analysis.Cultured cells were harvested in 150 µL of RIPA lysis buffer, and the protein concentration was measured.Proteins were denatured with 2× SDS at 95 °C for 5 min, and 20 μg of the denatured protein was loaded onto a 10% SDS-PAGE gel.The separated proteins were transferred to a polyvinylidene fluoride membrane (Amersham Hybond-P polyvinylidene fluoride membrane, GE HealthCare).The membranes were treated with a blocking reagent containing 5% BSA (Sigma-Aldrich) for 1 h at room temperature.Primary antibodies were applied in Can Get Signal Solution 1 (TOYOBO, Osaka, Japan) overnight at 4 °C and secondary antibodies in Can Get Signal Solution 2 (TOYOBO) for 1 h at room temperature.Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific) was used for visualization.Blots were scanned with ImageQuant™ LAS4000 mini4000 (Cytiva) and quantified using ImageJ (https:// imagej.nih.gov/ij/index.html).
The primary antibodies used were anti-TPPP3 [GTX33554] (GeneTex; euthanized by cervical dislocation at 2 months of age, and the forepaws were collected. Statistics and reproducibility.Data from the RT-qPCR, immunocytochemistry, Masson's trichrome staining, migration assay, chemotaxis assay, and immunohistochemistry assay are presented as mean ± standard deviation (SD).For the statistical comparison of RT-qPCR data, a parametrical two-tailed paired t-test or nonparametrical two-tailed Mann-Whitney U test was used.For the comparison of the Ki67-and αSMA-positive cell ratio of murine dermal fibrotic cells, αSMA-positive cell ratio of human Dupuytren's contracture-derived cells, migration and chemotaxis assays of RAW264.7 cells, dermal thickness, and number of myofibroblasts and CD206-positive mouse cells in immunocytochemistry, a non-parametrical two-tailed Mann-Whitney U test or one-way analysis of variance (ANOVA) with the Tukey or Tukey-Kramer multiple comparison test was used.Statistical analyses were performed using GraphPad Prism 9.4.0.Differences were considered statistically significant at p < 0.05.
Reporting summary.Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Fig. 1 TPPP3
Fig. 1 TPPP3 is expressed in the human palmar fascia and Dupuytren's contractures.a Haematoxylin and eosin staining (H&E) (left) and immunohistochemistry (right) of the human palmar fascia.TPPP3 expression was detectable in the peripheral connective tissue around the collagenous palmar fascia.b H&E staining (left) and immunohistochemistry (right) of human Dupuytren's contracture tissues.TPPP3 was strongly expressed in diseased cells in the nodule.c RT-qPCR results show the expression level of TPPP3 in 14 freshly isolated nodules of human Dupuytren's contractures (DD-1 to DD-14) (also see Supplementary Fig. 1) and in unaffected palmar fascia.TPPP3 expression was normalized to ACTB expression.The data are presented as the mean of three technical replicates.d Detection of TPPP3 expression in human Dupuytren's contracture samples (DD-11 and DD-13) using Western blotting.e Fluorescent immunohistochemistry of human palmar fascia (upper) and Dupuytren's contracture tissues (lower).TPPP3-expressing cells with fibroblast-like morphology on the surface of the palmar fascia did not co-express β-catenin or αSMA.Conversely, some TPPP3-expressing cells with vessel-like morphology co-expressed β-catenin and αSMA (upper).Most TPPP3-expressing cells in the nodule co-expressed β-catenin (upper) and the myofibroblast marker αSMA (lower).The white square area is enlarged in the upper right panel.Scale bars represent 50 µm (a, b, d).
demonstrated that tumour necrosis factor (TNF) treatment for palmar dermal fibroblasts derived from patients with Dupuytren's disease led to activation of Wnt/β-catenin signalling through GSK-3β phosphorylation and inhibition, resulting in upregulated COL1 and α-SMA expression.Meanwhile, activation of Wnt/β-catenin signalling by a GSK-3β inhibitor (SB-216763) did not induce COL1 and α-SMA expression.These findings suggest that the role of Wnt/β-catenin signalling in fibrosis might differ across cell types, biological species, and fibrosis models.Moreover, in addition to Wnt/β-catenin-mediated cell-autonomous myofibroblast differentiation, alternative mechanisms may exist for fibrosis development and progression.

Fig. 5
Fig. 5 Depletion of TGF-β signalling partially mitigates Wnt/β-catenin signalling-induced fibrosis in mice.a A schematic of the in vivo experiment on Tppp3-CreER T2 /β-catenin ex3 flox mice carrying a conditional deletion allele of TGF-β signalling (Tgfbr2 flox/flox ).At 1 month of age, the mice were injected with 1 mg of tamoxifen three times and analysed 1 month after injections.b Masson's trichrome staining of the palmar dermis of the paws.The upper panel shows a tamoxifen-treated Tppp3-CreER T2 /β-catenin ex3 flox mouse (Tgfbr2 wt), and the lower panel shows a tamoxifen-treated Tppp3-CreER T2 /β-catenin ex3 flox /Tgfbr2 flox/flox mouse (Tgfbr2-KO).Corn oil-treated Tppp3-CreER T2 /β-catenin ex3 flox mice served as the control group.Relative dermal thickness in each group was analysed using a one-way ANOVA with the Tukey-Kramer multiple comparison test.The mean thickness of the control group was set to 1. Compared with that in Tgfbr2 wt mice, dermal thickness was decreased significantly in Tgfbr2-KO mice (p = 0.0496).Biological samples encompassed control (n = 5), Tgfbr2 wt (n = 7), and Tgfbr2-KO (n = 5) groups.Dermal thickness was defined as the mean of six lesions measured per sample.c αSMA and CD31 immunohistochemistry of the paws.The upper panel shows a Tgfbr2 wt mouse, and the right panel shows a Tgfbr2-KO mouse.Myofibroblasts were identified as αSMA (green)-positive/CD31 (red)-negative cells.Relative myofibroblast counts in the dermis were analysed using a one-way ANOVA with the Tukey-Kramer multiple comparison test.The mean relative myofibroblast count of the control group was set to 1. Compared with that in Tgfbr2 wt mice, the relative number of myofibroblasts decreased significantly in Tgfbr2-KO mice (p = 0.0277).However, the relative number of myofibroblasts in Tgfbr2-KO mice was notably higher than that in control mice, indicating that the depletion of TGF-β signalling did not entirely rescue Wnt/β-catenin signalling-induced fibrosis.Biological samples included control (n = 4), Tgfbr2 wt (n = 6), and Tgfbr2-KO (n = 5) groups.Two different lesions were analysed per sample.Scale bars represent 50 µm (b, c).The data are presented as the mean ± SD (b, c).

Fig. 6
Fig.6Dermal fibrotic cells express Cxcl14 via Wnt signalling activation.a The expression (RT-qPCR) of M2 macrophage-associated chemokines.Isolated dermal fibrotic cells from the paws of doxycycline (Dox)-inducible β-catenin mice (Fig.3d) were divided into Dox-on and Dox-off groups.The expression level in the Dox-off group was set to 1.The mean ± SD of seven biological samples (three technical replicates per sample) are shown (two-tailed paired t test).b Immunohistochemistry of Cxcl14 in dermal fibrotic lesions of the paw in Tppp3-CreER T2 /β-catenin ex3 flox (left) and control mice (Tppp3-CreER T2 /Rosa26-stop-tdTomato) (right).Samples were obtained 1 month after three tamoxifen injections of 1 mg.Marked Cxcl14 expression was detected in dermal fibrotic lesions (left).c Fluorescent immunohistochemistry was employed to visualize the distribution of Cxcl14 (green) and tdTomato (red) in the context of dermal fibrosis in the paw of Tppp3-CreER T2 /Rosa26-stop-tdTomato/Rosa26-stop-rtTA/Col1a1::TetO-β-catenin mice (upper panel: Dox-off mice; lower panel: Dox-on mice).Dox-treated tdTomato-positive cells (β-catenin-expressing Tppp3-lineage cells) in the dermis co-expressed Cxcl14 (indicated by white arrowheads).The white square area is enlarged in the upper right panel.d Gene expression analysis by RT-qPCR of recombinant Cxcl14-treated (10 nM) murine macrophages (RAW264.7).Cxcl14 treatment did not induce M2 macrophage differentiation.The mean ± SD values of seven independent experiments (three technical replicates per sample) are shown (two-tailed Mann-Whitney U test). e Chemotaxis assay of recombinant Cxcl14-treated (10 nM) macrophages.Compared to the control group (upper panel), Cxcl14 treatment (lower panel) showed significantly increased cell infiltration (p = 0.0174).The mean ± SD values of four independent experiments (three different lesions per experiment) are shown (two-tailed Mann-Whitney U test).f Migration assay of recombinant Cxcl14-treated (10 nM) macrophages.Compared to the control (left), the Cxcl14 treatment (right) showed significantly increased cell migration after 30 h (p = 0.0101).The mean ± SD of three independent experiments (three different lesions per experiment) are shown (two-tailed Mann-Whitney U test).g Chemotaxis assay of control IgG and anti-Cxcl14 antibody-treated macrophages.Compared to the control medium, the conditioned medium significantly increased cell infiltration (p < 0.0001).Compared to the conditioned medium alone, control IgG treatment did not yield a statistically significant reduction in cell infiltration (p = 0.6859).Conversely, anti-Cxcl14 antibody treatment resulted in a significant decrease in cell infiltration (p < 0.0001).The mean ± SD values of three independent experiments (three different lesions per experiment) are shown (oneway ANOVA with Tukey-Kramer multiple comparison test).CM; conditioned medium.h Fluorescent immunohistochemistry of Cxcl14 (green) and CD206 (red) in the context of dermal fibrosis in the paw of Tppp3-CreER T2 /β-catenin ex3 flox mice (1 month after tamoxifen injection).CD206-expressing M2 macrophages were polarized around Cxcl14-expressing cells.Some M2 macrophages co-expressed Cxcl14 (indicated by white arrowheads).The white square area is enlarged in the upper right panel.i Cxcl14 expression (RT-qPCR) in recombinant TGF-β1 (10 ng/mL)-treated murine dermal fibrotic cells derived from Tppp3-CreER T2 /β-catenin ex3 flox mice.TGF-β1 treatment significantly upregulated CXCL14 expression (p = 0.0317).The mean ± SD of six independent biological samples (three technical replicates per sample) are shown (two-tailed Mann-Whitney U test).Scale bars represent 20 µm (b), 50 µm (c, f, g), or 200 µm (e).

Fig. 7
Fig. 7 Neutralization of Cxcl14 partially suppresses Wnt/β-catenin-induced dermal fibrosis by inhibiting M2 macrophage infiltration in mice.a A schematic of the experimental protocol of anti-Cxcl14 neutralizing antibody treatment for Tppp3-CreER T2 /β-catenin ex3 flox mice.Mice were treated with 15 μg of an anti-Cxcl14 neutralizing antibody or control IgG twice a week for 1 month.b Fluorescent immunohistochemistry of CD206 (red) in the paws.The left panel shows control IgG treatment, and the right shows anti-Cxcl14 neutralizing antibody treatment after tamoxifen injections.Dotted lines indicate the border of the dermis.The relative CD206-positive cell count in each group was analysed (one-way ANOVA with the Tukey multiple comparison test).Biological samples comprised corn oil (n = 4), tamoxifen + control IgG (n = 4), and tamoxifen + Cxcl14 antibody (n = 4) groups.Two different lesions were analysed per sample.c Masson's trichrome staining of the dermal fibrotic lesion of the paws.The left panel shows control IgG treatment, and the right shows anti-Cxcl14 neutralizing antibody treatment.Relative dermal thickness in each group was analysed (one-way ANOVA with the Tukey-Kramer multiple comparison test).Compared to the control treatment (corn oil), control IgG treatment did not exhibit any protective effect against Wnt/β-catenin-induced fibrosis (p = 0.0086), whereas anti-Cxcl14 neutralizing antibody treatment demonstrated a discernible protective effect in this context (p = 0.2978).The mean thickness of the control group was set to 1. Biological samples comprised corn oil (n = 5), tamoxifen + control IgG (n = 8), and tamoxifen + Cxcl14 antibody (n = 8) groups.Dermal thickness was defined as the mean of four lesions measured per sample.d Fluorescent immunohistochemistry of αSMA (green) and CD31 (red) in the dermis of the paws.Left: control IgG treatment after tamoxifen injections; lower: anti-Cxcl14 neutralizing antibody treatment after tamoxifen injections.Myofibroblasts were defined as αSMA-positive/CD31-negative cells.Relative myofibroblast counts in the dermis were analysed (one-way ANOVA with the Tukey multiple comparison test).Compared with the control IgG treatment, anti-Cxcl14 antibody treatment significantly decreased the number of myofibroblasts in the dermis (p = 0.0202).Biological samples included corn oil (n = 4), tamoxifen + control IgG (n = 4), and tamoxifen + Cxcl14 antibody (n = 4) groups.Two different lesions were analysed per sample.Scale bars represent 50 µm (b, c, d).The data are presented as the mean ± SD (b, c, d).

Fig. 8
Fig. 8 Schematic representation of the cell-and non-cell-autonomous fibrotic program induced by Wnt/β-catenin signalling activation in Tppp3positive cells.